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HEPES buffer – or (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid) – is a biological buffer used in cell culture. In chemistry, a buffer is a solution that contains an acid and a base or salt. Its main purpose is to maintain the concentration of hydrogen ions at a level that’s ideal for specific purposes, such as a bacterial culture. 

How Does HEPES Buffer Work?

Just like other types of chemical buffers, HEPES buffer maintains the ion concentration of a solution at a level that’s ideal for a certain purpose. The pH level is maintained in this manner. Specifically, HEPES buffer is used to maintain the ideal conditions for cell culture. Therefore, it’s classed as a biological buffer and is best suited for microbiology and histology. It’s also useful in medicine as part of diagnostic laboratory procedures.

This buffer is classified as a zwitterionic sulphonic acid buffering agent with the IUPAC chemical name 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Its chemical formula can be written as C8H18N2O4S, while its structural formula is written as:


HEPES buffer has the following properties:

  • Molar mass: 238.3012 g/mol
  • Melting point: 234 °C
  • Soluble in: Water
  • Classification: Sulphonic acid

HEPES buffer is one of the twenty Good’s buffers. These are named after Norman Good who, together with his colleagues, prepared and tested these buffers between 1966 and 1980. Before the work of Good and his colleagues, biologists didn’t have access to a wide range of appropriate buffers; many were very reactive, toxic, and inefficient.

All Good’s buffers have the following properties:

  • Highly soluble in water
  • Low level of permeability to cell membranes
  • The dissociation constants are stable and consistent
  • Low metal chelating capability
  • High chemical stability
  • Not very absorptive of UV spectra

The complete list of Good’s buffers and their properties includes:

Many biological buffers formerly used by researchers were reactive and inefficient, making them unfit for cell culture. HEPES is now widely used in culturing cells, particularly in the laboratory setting. Compared to bicarbonate buffers, HEPES is better at maintaining the physiological pH of cells despite the changes in the level of carbon dioxide produced during aerobic respiration.

However, unlike bicarbonate buffers, HEPES buffer is very sensitive to light, producing hydrogen peroxide when exposed to it. Hydrogen peroxide is toxic to cells because of its oxidising property. Therefore, it’s crucial to store and use this buffer in darkness as much as possible. 

What is the pH of HEPES Buffer?

HEPES buffer’s useful pH ranges are from 2.5 to 3.5 for acidic solutions and from 6.8 to 8.2 for basic solutions. That said, depending on the type of cells to be cultured, such as eukaryotic, prokaryotic, unicellular organisms, or tissue cells, HEPES buffer can be prepared to whatever the ideal pH is. 

Tissue cells and unicellular organisms have an ideal narrow pH range for growth. Most bacteria, for example, ideally grow in a culture medium that has a neutral or near neutral pH of between 6.5 and 7.0. On the other hand, liver tissue cells have the ideal culture medium pH range of between 7.3 and 7.45. 

HEPES buffer has two pKa constants, which refer to its dissociation constants as ions:

  • pKa1 (25 °C) = 3
  • pKa2 (25 °C) = 7.5

This buffer has a negligible metal ion binding property, which makes it an ideal buffer for enzymes that could be inhibited by metal chelation, or the bonding of ions and molecules to metal ions. Chelation involves the presence or formation of two or more separate coordinate bonds between a multiple bonded ligand and a single atom.

All types of cells, whether microbial cells or tissue cells, produce waste products that are toxic to the cells itself. These waste products change the pH, chemical compositions, and other properties of the culture medium. The pH is very important in terms of optimal conditions that allow cell growth. Maintaining the pH within a narrow range is crucial for culturing cells under ideal conditions.

How is HEPES Buffer Used in Cell Culture?

Culturing cells in a laboratory setting has various purposes. It can be for diagnosing certain types of diseases, genetic profiling, identifying microbe species, testing the efficacy of antibiotics, growing replacement skin tissues for skin graft, experimenting on cancer cells, and extracting biomolecules.

Culturing cells in tissue culture plates.

Living cells undergo metabolic processes that have three basic requirements: a source of nutrition and energy, a means to expel waste products, and an oxygen or substitute electron acceptor. Anaerobic organisms also undergo respiration, but they use other types of electron acceptors in the electron transport chain, such as the following:

  • Nitrate (NO3−)
  • Fumarate
  • Sulfate (SO42−)
  • Sulfur (S)

HEPES buffer is added to the culture medium at the start of the cell culture process. Its main role is to maintain the pH level of the culture medium as carbon dioxide accumulates because of the respiration of the cells. The water in the culture medium, like agar, reacts with carbon dioxide to form carbonic acid, which is similar to the way acid rain is formed in the atmosphere:

The amount of HEPES buffer can be adjusted depending on the ideal pH range of the cells being cultured. All cells have limitations in terms of growth. These include the space or volume occupied by the culture medium, the amount of nutrients and energy sources, such as glucose, the amount of waste products, the temperature, and, finally, the pH.

As we touched on earlier, cell culture media that contain HEPES buffer must not be exposed to light for a prolonged period of time in order to prevent the formation of hydrogen peroxide. Cells will die if hydrogen peroxide accumulates in the solution.

In most microbiology laboratories, the basic laboratory equipment needed for cell culture includes:

  • Cell culture hood or biosafety cabinet
  • Incubator (humid CO2 incubator)
  • Water bath
  • Centrifuge
  • Refrigerator and freezer
  • Cell counter
  • Inverted microscope
  • Liquid nitrogen (N2) freezer or cryostorage container
  • Steriliser, such as an autoclave

Additional safety equipment, such as complete sets of PPE, may also be required depending on the biohazard classification of the cells being cultured. For most laboratories, basic safety gears are used, such as rubber gloves and goggles.

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